Process for producing vitamin B12 from hydrogen-metabolizing methane bacterium

ABSTRACT

Mesophilic methane bacteria obtained from digested sludge are acclimatized in an H 2 /CO 2  medium and the acclimatized methane bacteria are grown on a support inclusive of inorganic nutritional salts of trace metal elements by using an immobilized bed bioreactor. The grown bacteria are allowed to metabolize a mixed gas, prepared by adding hydrogen to at least one of coal gas and biogas, into methane and at the same time, cobalamin contained in the fermented liquor is recovered as an extracellular product in the form of cyanocobalamin by using potassium cyanide to thereby produce vitamin B 12  efficiently in a high content and high yield.

FIELD OF THE INVENTION

The invention of this application relates to the method for producing vitamin B₁₂.

It has been known that cobalamin, that is, vitamin B₁₂ analogues can be chemically synthesized with difficulty and biosynthesized from bacteria or actinomycetes, and yeast, fungi, plants and animals have no ability to biosynthesizing vitamin B₁₂. A method of extracting vitamin B₁₂ from cell bodies of propionibacteria using methanol as a substrate is being put to industrial use. Although such vitamin B₁₂ is primarily utilized as drugs, there are increased demands for the development of functional foods and for health-maintaining foods to which vitamin B₁₂ is added. And therefore a development of less the cost vitamin B₁₂ is desired in recent years.

BACKGROUND ART

Heretofore, in the production of vitamin B₁₂ from propionibacteria, methanol is used as a substrate and the growth of these bacteria is very slow because these bacteria are facultative anaerobe. Also, because propionic acid is transformed into acetic acid, and the acetic acid caused growth inhibition. So that the density of cell bodies in fermented liquor is not increased and the amount of vitamin B₁₂ that can be extracted from the cell body is the order of 1 mg/L, showing that the conventional process has low productivity. And Shiro Nagahisa et al. (Seibutsu-kogaku Kaishi, 76(6), 447–455, 1998) used an immobilized bed bioreactor of methane bacteria wherein methanol is used as a substrate to compare the yield of vitamin B₁₂ of methane bacteria with that of propionibacteria and obtained vitamin B₁₂ from methane bacteria in a yield 10 times higher than that obtained from propionibacteria. However, the resulting solution contains, as its major component, collinoid having small cobalamin types. In this case, there is the problem that the recovery of vitamin B₁₂ is not improved because a process of separation from methanol is complicated and this method does not bring about a reduction in production cost since the content of vitamin B₁₂ is small.

DISCLOSURE OF THE INVENTION

In view of this situation, it is an object of the invention of this application solves the prior art problems as aforementioned and to provide a new method ensuring that vitamin B₁₂ can be efficiently produced in a high content and high yield.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a view illustrating an increase in the density of methane bacteria.

FIG. 2 shows a view illustrating the ratio of extracellular vitamin B₁₂ to intracellular vitamin B₁₂.

FIG. 3 shows a view illustrating the relation between Co/Fe ratio and the density of cyanocobalamin.

FIG. 4 shows an example of the structure of a bioreactor.

FIG. 5 shows another example of the structure of a bioreactor.

FIG. 6 shows a view illustrating the relation between the activity of methane bacteria and the content of vitamin B₁₂ in an immobilized bed fermentor.

It is to be noted that the symbols in FIG. 4 and FIG. 5 represent the following parts.

1. Porous ceramics

2. Reactor

3. Support packed vessel

4. Negative electrode

5. Diffusing nozzle

6. Electromagnetic valve

7. Gas holder

8. Biogas or coal gas feed pipe

9. Oxygen discharge section

10. Positive electrode

11. Electromagnetic valve

12. Electromagnetic valve

13. Pressure detection controller

14. Check valve

15. Drain valve

16. Drain valve

17. Electrolysis apparatus

18. Ultra-filtration membrane unit

19. Pressure control electromagnetic valve

20. Gas density detection controller

21. Liquid supply nozzle for a medium

BEST MODE FOR CARRYING OUT THE INVENTION

The invention of this application of this case has been made to solve the aforementioned problem and, first, provides a method for producing vitamin B₁₂, the method comprising acclimatizing mesophilic methane bacteria obtained from digested sludge in a H₂/CO₂ medium, growing the acclimatized methane bacteria on a support inclusive of inorganic nutritional salts of trace metal elements by using an immobilized bed bioreactor, allowing the bacteria to metabolize a mixed gas, prepared by adding hydrogen to at least one of coal gas and biogas, into methane and, at the same time, recovering cobalamin contained in the fermented liquor as an extracellular product in the form of cyanocobalamin by using potassium cyanide.

Second, the invention of this application provides a method for producing vitamin B₁₂, the method comprising acclimatizing mesophilic methane bacteria obtained from digested sludge in a H₂/CO₂ medium, growing the acclimatized methane bacteria in a suspension culture reactor combined with a ultra-filtration membrane and containing trace metal elements, recovering extracellular cobalamin contained in the solution penetrated through the ultra-filtration membrane as cyanocobalamin by using potassium cyanide and allowing the methane bacteria to return to the culture solution from the ultra-filtration membrane and culturing the methane bacteria to metabolize a mixed gas prepared by adding hydrogen to at least one of coal gas and biogas.

Also, the invention of the patent application of this case provides, third, in the above method, a method in which the concentration of the trace metals is made to be 10 to 1000 times higher than that of the standard composition and, fourth, a method in which the ratio of Co/Fe among the concentrations of the trace metal salts in the medium of hydrogen-metabolizing methane bacteria is made to be 10 to 100 times higher than the standard composition ratio.

Further, the invention provides, fifth, a method for fermenting methane as the method of adding hydrogen to coal gas and biogas, the method comprising placing a negative electrode portion at the upper position and a positive electrode portion at the lower position through a porous ceramics on the bottom of an immobilized bed or suspension type bioreactor, applying D.C. voltage to electrolyze water in a culture solution thereby generating hydrogen, which is utilized in a trace element-enriched support immobilized bed or complex type methane fermenting bioreactor, and emitting oxygen into the atmosphere and, sixth, a method of reforming gas, the method comprising applying D.C. voltage to the residue obtained by extracting cobalamin, which is an extracellular product, as cyanocobalamin by using potassium cyanide to perform electrolysis and supplying hydrogen obtained by the above extraction to coal gas and biogas containing carbon monoxide and carbon dioxide to perform methanation by using a membrane-combined bioreactor or an immobilized bed reactor.

The invention of this application has been completed taking the opportunity of acquiring the following findings and also based on these findings. Specifically, the inventors of the patent application of this case have found that in an experiment made to try converting CO₂ (about 40%) in biogas into CH₄ (methanation) by adding H₂ in the development of a support inclusive of trace elements/inorganic nutrient salts (JP-A-9-140181 and JP-A-10-327850), it is clarified from the calculation of the material balance of vitamin B₁₂ that extracellular dilution of vitamin B₁₂ present in a culture solution is caused and vitamin B₁₂ existing in a cell body is only 30% when the amount of vitamin B₁₂ is 70% of the total amount in the experiment of methanation (4H₂+CO₂→CH₄ +2H₂O), although it is said in the conventional reports that a large part of vitamin B₁₂ exists in a cell body in the case of using bacteria other than methane-generating bacteria and the methane bacteria obtained by selectively culturing mesophilic methane bacteria by acclimatizing culture using, as a substrate, a mixed gas of hydrogen and carbon dioxide is capable of extracting highly concentrated cobalamin (analogues of vitamin B₁₂) as cyanocobalamin from the fermented liquor.

Firstly, in the method of the invention of the patent application of this case, basically;

1) mesophilic methane bacteria obtained from digested sludge are acclimatized in a H₂/CO₂ medium;

2) the acclimatized methane bacteria are grown using an immobilized bed bioreactor on a support inclusive of inorganic nutrient salts of trace metal elements;

3) a mixed gas prepared by adding hydrogen to coal gas and biogas is metabolized into methane and at the same time;

4) cobalamin present in the fermented liquor as an extracellular product as cyanocobalamin, namely, vitamin B₁₂ by using potassium cyanide.

In this method, for example, it is effective to raise the concentration of the trace metals 10 to 1000 times higher than that used conventionally in the above inclusive support.

In, for example, suspension culture in a membrane-combined type bioreactor equipped with an ultra-filtration membrane, it is effective to raise each concentration of the trace metal salts and nutrient salts 10 to 1000 times higher than that used conventionally. As to this ultra-filtration membrane-combined suspension culture, in this invention, the aforementioned method for producing vitamin B₁₂ is provided resultantly, wherein mesophilic methane bacteria obtained from digested sludge are acclimatized in a H₂/CO₂ medium, the acclimatized methane bacteria are grown in the ultra-filtration membrane-combined suspension culture reactor containing trace metal elements, extracellular cobalamin contained in the solution penetrated through the ultra-filtration membrane is recovered as cyanocobalamin by using potassium cyanide and the methane bacteria are allowed to return to the culture solution from the ultra-filtration membrane and cultured to metabolize a mixed gas prepared by adding hydrogen to at least one of coal gas and biogas.

In all of the aforementioned methods in the present invention, the concentration of cobalamin in the culture salts can be increased by such a partial alteration of a medium that particularly the ratio of Co/Fe is 10 to 100 times higher. Therefore, the invention reforms coal gas and biogas which are obtained by methanation for converting 100% of coal gas and biogas containing a large amount of CO₂ and CO into CH₄, and at the same time, cobalamin is converted into cyanocobalamin (vitamin B₁₂) by using potassium cyanide, which is recovered from the culture solution. The recovery of vitamin B₁₂ is very simplified as compared with a conventional method of the recovery from cell bodies and the concentration of vitamin B₁₂ is 50 to 100 times higher than that obtained using propionibacteria. Also the recovery from this culture medium reaches 2 to 10 times that from methanol-metabolizing methane bacteria (Shiro Nagai et al. 1996. Seibutsu-kogaku Kaishi, 74(6), 447–455), resulting in that the production cost of vitamin B₁₂ can be remarkable reduced.

Next, the invention of this application will be further explained according to the drawings attached.

FIG. 1 is a view showing an increase in the density of cell bodies of methane bacteria when, using a gas containing 80% of hydrogen and 20% of carbon dioxide as the substrate, methane bacteria which are sufficiently acclimatized using a liquid type standard synthetic medium are continuously cultured at the same medium temperature (35° C. to 36° C.) using a support inclusive of inorganic nutrient salts in which each concentration of the nutrient salts and trace metal salts is raised 10 to 1000 times higher than that used in conventional methods (JP-A-9-140181 and JP-A-10-327850, trace elements/inorganic nutrient salts diffusion type cell body culture support). When the density of cell bodies of methane bacteria was 26 g-dry/L, the concentration of vitamin B₁₂ reached 185 mg/L. This concentration was 50 to 100 times that obtained in the case of using propionibacteria.

Also, in FIG. 2, 80% of hydrogen and 20% of carbon dioxide were blown as a substrate into a synthetic medium having a trace metal concentration 10 times higher than that of a conventional method to examine the ratio of intracellular vitamin B₁₂ to extracellular vitamin B₁₂ in the suspension culture solution to find that each proportion was 30% and 70%. So it is considered that the separation of vitamin B₁₂ eluted extracellularly by using a support inclusive of trace elements/nutrient salts rather enables efficient production.

Then, FIG. 3 shows the density of cyanocobalamin when operating a bioreactor using, as parameters, the ratio of cobalt to iron which are the trace elements in the support inclusive of the trace elements/nutrient salts by using, as the substrate, a mixed gas having a carbon dioxide concentration of 40% to 60% and a hydrogen concentration of 60%. Cobalamin is an analogue of vitamin B₁₂ and it is understood that cobalamin strongly requires cobalt.

In FIG. 4, as a method of adding hydrogen to carbon dioxide gas and biogas, a porous ceramics 1 is placed on the bottom of a reactor 2 in a biogas reactor, a metal electrode (titanium and iron) corresponding to a positive electrode 10 is placed under the porous ceramics 1 in a solution at the lower most portion of the reactor, an electrode plate of a negative electrode 4 made of platinum or titanium is placed just above a diffusing nozzle 6 disposed just above the porous ceramics 1 and a voltage of 20 to 100 V is applied. Hydrogen is thereby generated on the plate of the negative electrode 4. Although oxygen is generated on the electrode plate of the positive electrode 10, it is diffused into air by means of a blower B2 by disposing an oxygen-emitting section 9. If the size of the clearance of the porous ceramics 1 is designed to be as small as 1/20 to 1/100 of the diameter of the generated oxygen cell and the pressure is always kept higher than the atmospheric pressure by using a blower, oxygen generated by electrolysis never penetrates into the reactor 2 through the porous ceramics 1 and an absolutely anaerobic condition in the bioreactor can be kept. A support packed vessel 3 is incorporated into the reactor 2 such that a support inclusive of trace elements is disposed as an immobilized bed. The volume of the support packed vessel is designed to be 30 to 60% of the effective volume to fill it. Coal gas and biogas are introduced from a biogas or coal gas feed pipe 8 according to the indication of a pressure detection controller 13. These gases are circulated by the operation of a blower B1 and by an open-operation of electromagnetic valves 11 and 12. When the concentration of carbon dioxide is decreased and the concentration of CH₄ is increased, an electromagnetic valve 6 is opened according to the indication of a gas temperature detection controller designated as the symbol 20 to feed methane to a gasholder 7. When the pressure in the bioreactor 2 is dropped, the electromagnetic valves 6 and 12 are opened to supply gas newly from the biogas or coal gas feed pipe 8.

In FIG. 5, the fermented liquor containing cobalamin is decomposed in an electrolysis apparatus 17 and only hydrogen is supplied to the bioreactor 2. Methane bacteria living in the immobilized bed of the support packed vessel 3 run methanation to convert carbon dioxide, fed from the biogas or coal gas feed pipe 8, into methane by hydrogen supplied from the electrolysis apparatus 17. When the concentration of methane in the bioreactor rises and reaches close to 100%, the valve 6 is opened by the gas detection controller 20 to feed methane to the gasholder 7. When the pressure in the bioreactor is dropped, the electromagnetic valves 6 and 12 are put into a state of closed-operation and gas is newly supplied from the biogas or coal gas feed pipe 8 by an open-operation of the electromagnetic valve 11. In this manner, both FIG. 4 and FIG. 5 show a semi-continuous operation when gas is used as a substrate. On the other hand, because the concentration of cobalamin rises in the liquor, the concentration is always inspected and the liquor is withdrawn in a fixed amount by the operation of drain valves designated as the symbols 15 and 16 respectively when the concentration reaches a maximum to recover vitamin B₁₂ from this liquor. As to the withdrawal of the liquor, it is withdrawn using the drain valve 15 or 16 on the premise that new gas is supplied, provided that these operational procedures are carried out so as to prevent the intrusion of oxygen into the bioreactor 2. Thereafter, a medium solution is supplied from a medium solution supply nozzle 21 as shown in FIG. 4 and FIG. 5 by using a pump.

These methods and apparatuses illustrated in FIG. 4 and FIG. 5 are useful in the practice of any one of the first to fourth methods of this invention.

Next, examples of this invention will be explained.

EXAMPLES

<1> In an experiment using batch suspension culture wherein the concentration of cobalt chloride among the concentration of trace elements which concentration was 10 times higher than that of conventional method was raised, the concentration of vitamin B₁₂ in the culture solution was 25.2 mg/L after 48 hours in the case of using, as a substrate, a synthetic gas containing 20% of CO₂ and 80% of H₂. The conventional bibliographic value of the concentration of vitamin B₁₂ obtained from propionibacteria was 0.5 to 1 mg/L. It was therefore confirmed that the method of this invention had high yield and cyanocobalamin eluted extracellularly so that it was recovered with ease. <2> A solution having a concentration 1000 times higher than that of a conventional method was confined in a support inclusive of a trace element/nutrient fixed to the support and then the support was packed in an immobilized bed bioreactor having a volume of 5 L. Hydrogen generated by a hydrolysis process in a culture solution was continuously supplied to biogas consisting of 40% of CO₂ and 60% of CH₄ to carry out a continuous operation for reforming the biogas, whereby 98 to 99% of the biogas could be converted into CH₄. Also, at the same time, the concentration of vitamin B₁₂ in the culture solution reached 180 mg/L. This concentration was 50 to 100 times that obtained using proplonibacteria and the production rate was also 25 to 50 times that obtained using propionibacteria.

From the above, the specific action and marked results of the examples are summarized as follows:

1. Vitamin B₁₂ is recovered from a culture solution having enriched trace metal salts but not from a cell body itself. Therefore, cell bodies can be kept in a high density and cobalamin, which is an extracellular product, can be recovered in by far much amount as compared with the case of using a conventional method and the yield of vitamin B₁₂ can be raised.

2. Hydrogen can be supplied from a solution itself contained in the bioreactor by an electrolysis process. This makes it possible to reform biogas and coal gas and to attain high energization of these gases.

3. In the method of recovering vitamin B₁₂, the uses of potassium cyanide simplify the recovery of vitamin B₁₂ as cyanocobalamin, decreasing the production cost of vitamin B₁₂ more greatly.

<3> A solution having a trace element concentration 1000 times higher than that of a conventional method was confined in a support inclusive of nutrient salts and then the support was packed in an immobilized bed bioreactor having a volume of 5 L. A continuous sulfurization experiment was conducted in a medium having a concentration 100 times higher than that of a conventional method in two conditions of mean residence time (HRT) of 3 days and 6 days. At this time, hydrogen generated by electrolyzing a supernatant liquor side medium was continuously supplied to biogas consisting of 40% of CO₂ and 60% of CH₄ to carry out a continuous operation for reforming the biogas for 30 days. The results are shown in FIG. 6. First 9 days corresponds to 3 times the HRT required for start-up. Considering that the system is in an unsteady state during this period, the production rate of methane and cyanocobalamin vitamin B₁₂ (perfect type) were measured since 9th day till 30th day. As a result, in the case where HRT was 6 days, the perfect type vitamin B₁₂ was kept in a concentration of about 37.5 mg/L and the production rate of methane was 11.5 L/L/h. When the rate of the production of the perfect type vitamin B₁₂ is compared taking HRT into account, it is 110 to 160 times that of the production from propionibacteria. As compared with the results of TAPPAN et al. (1987: Applied Microbiology and Biotechnology, 26: 511–516), the results of FIG. 6 show that a twenty-fold increase in the production rate of the perfect type vitamin B₁₂ is attained.

Also, the density of methane bacteria in the experiment shown in FIG. 6 reaches 40 g/L. It is therefore clarified that the perfect type vitamin B₁₂ is eluted in a large amount from methane bacteria, offering possibilities of stabilizing the industrial production of vitamin B₁₂ and of achieving low production cost.

INDUSTRIAL APPLICABILITY

As mentioned above in detail, the invention of ensures that vitamin B₁₂ can be produced in a high content and high yield by far more efficiently than in the case of conventional production. 

1. A method for producing vitamin B₁₂, the method comprising acclimatizing mesophilic methane bacteria obtained from digested sludge in a H₂/CO₂ medium, growing the acclimatized methane bacteria on a support inclusive of inorganic nutritional salts of trace metal elements by using an immobilized bed bioreactor, allowing the bacteria to metabolize a mixed gas, prepared by adding hydrogen to at least one of coal gas and biogas, into methane and, at the same time, recovering cobalamin contained in the fermented liquor as an extracellular product in the form of cyanocobalamin by using potassium cyanide.
 2. A method for producing vitamin B₁₂, the method comprising acclimatizing mesophilic methane bacteria obtained from digested sludge in a H₂/CO₂ medium, growing the acclimatized methane bacteria in a suspension culture reactor combined with a ultra-filtration membrane and containing trace metal elements, recovering extracellular cobalamin contained in the solution penetrated through the ultra-filtration membrane as cyanocobalamin by using potassium cyanide and allowing the methane bacteria to return to the culture solution from the ultra-filtration membrane and culturing the methane bacteria to metabolize a mixed gas prepared by adding hydrogen to at least one of coal gas and biogas.
 3. A method according to claim 1, wherein the ratio of Co/Fe among the concentrations of the trace metal salts in the medium of hydrogen-metabolizing methane bacteria is in a range of ⅕ to 1/20.
 4. A method according to claim 2, wherein the ratio of Co/Fe among the concentrations of the trace metal salts in the medium of hydrogen-metabolizing methane bacteria is in a range of ⅕ to 1/20.
 5. A method according to claim 1, wherein the trace metals are at least one member selected from the group consisting of: MgCl₂.6H₂O in a range of 4100 μg/L to 410000 μg/L; MnCl₂.4H₂O in a range of 500 μg/L to 50000 μg/L; FeCl₃.4H₂O in a range of 500 μg/L to 50000 μg/L; NiCl₂.6H₂O in a range of 120 μg/L to 12000 μg/L; ZnSO₄.7H₂O in a range of 100 μg/L to 10000 μg/L; CaCl₂.2H₂O in a range of 100 μg/L to 10000 μg/L; CoCl₂.6H₂O in a range of 100 μg/L to 10000 μg/L; Na₂SeO₃ in a range of 80 μg/L to 8000 μg/L; Na₂MoO₄.7H₂O in a range of 24 μg/L to 2400 μg/L; CuSO₄.5H₂O in a range of 10 μg/L to 1000 μg/L; AlK(SO₄)₂ in a range of 10 μg/L to 1000 μg/L; H₃BO₄ in a range of 18 μg/L to 1800 μg/L; and NaWO₄.2H₂O in a range of 10 μg/L to 1000 μg/L.
 6. A method according to claim 2, wherein the trace metals are at least one member selected from the group consisting of: MgCl₂.6H₂O in a range of 4100 μg/L to 410000 μg/L; MnCl₂.4H₂O in a range of 500 μg/L to 50000 μg/L; FeCl₃.4H₂O in a range of 500 μg/L to 50000 μg/L; NiCl₂.6H₂O in a range of 120 μg/L to 12000 μg/L; ZnSO₄.7H₂O in a range of 100 μg/L to 10000 μg/L; CaCl₂.2H₂O in a range of 100 μg/L to 10000 μg/L; CoCl₂.6H₂O in a range of 100 μg/L to 10000 μg/L; Na₂SeO₃ in a range of 80 μg/L to 8000 μg/L; Na₂MoO₄.7H₂O in a range of 24 μg/L to 2400 μg/L; CuSO₄.5H₂O in a range of 10 μg/L to 1000 μg/L; AlK(SO₄)₂ in a range of 10 μg/L to 1000 μg/L; H₃BO₄ in a range of 18 μg/L to 1800 μg/L; and NaWO₄.2H₂O in a range of 10 μg/L to 1000 μg/L. 